The present invention relates to the field of molecular virology and more particularly to nucleic acid probes for detection of viruses.
Viral infections and virus-related diseases are significant medical problems which cause personal pain and suffering as well as great economic hardship to individuals, families, and society. Of particular concern are retroviruses, including human immunodeficiency virus (HIV), the virus causing acquired immunodeficiency syndrome or AIDS.
Retroviruses invade host organisms, such as humans, by attaching to the exterior surface of a host cell and introducing their genetic material, retroviral ribonucleic acid (RNA), into the host cell's cytoplasm. The viral enzyme reverse transcriptase, which is packaged in the virus, generates a DNA copy of the viral genome from the viral RNA. This DNA copy usually integrates into the host cell genome where it is chemically indistinguishable from host cellular DNA. The integrated viral genome is copied by host cell replicative mechanisms and transmitted to the cellular progeny of the infected cell. In addition to replicating the viral DNA, the cellular machinery of the infected host cell transcribes viral DNA into messenger RNA (mRNA) and further translates the viral mRNA into enzymatically active viral proteins. The viral proteins thus formed act within the host cell to produce additional viable and infectious virus particles.
The retrovirus family is characterized by the presence of reverse transcriptase in the virions. This family is composed of several genera including oncovirus, spumavirus, and lentivirus. The typical retrovirus genome is short, generally less than 10 kilobases in length, and simple, usually consisting of only a few genes. The major genes of the retroviral genome are gag, pol, and env, as shown in FIG. 1, which encode structural capsid proteins, reverse transcriptase and related replication proteins, and viral extra-cellular envelope protein components, respectively.
The Lentivirus genus includes HIV and related viruses, some causing immunodeficiency-like disease. Lentiviruses are often distinguished by their ability to cause slowly developing diseases characterized by a long incubation period and protracted course of illness. These viruses often latently infect monocytes and macrophages and then spread to other cells. Lentiviruses generally have a bar-shaped rather than a spherical nucleoid. The genome of lentiviruses normally use a tRNA.sup.lys as a primer for negative strand synthesis rather than tRNA.sup.pro , which is used by most other infectious mammalian retroviruses except spumaviruses and mouse mammary tumor viruses. The reverse transcriptases of lentiviruses are magnesium dependent while mammalian C type viruses (oncoviruses) prefer manganese. Also, lentiviruses are known for their genome complexity in that the genome contains additional genes such as the additional regulatory genes.
Lentiviruses in general, and HIV in particular, are known to mutate frequently and to have tremendous evolutionary potential. In addition, numerous variants of HIV and related immunodeficiency-like lentiviruses probably exist that have not been isolated, studied, or characterized. Such unidentified strains may be responsible for clinical immunodeficiency or other disorders not attributable to currently known lentiviruses.
One of the major problems in the diagnosis of such immunodeficiency disorders is the identification and characterization of the causative agent, the lentiviral genome, which constitutes only a very small fraction of the total genetic material in the patient's body. It is extremely difficult to distinguish such a minute amount of viral related genetic material from the great bulk of normal cellular genetic material contained in the host cell.
One recently developed approach for the detection of retroviral genomes employs the polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis. PCR has emerged as a powerful and sensitive procedure for the amplification of specific DNA sequences (given proper oligonucleotides for use as primers for the reaction), and as such is a valuable diagnostic tool for the identification and characterization of viral diseases. PCR technology requires pairs of dissimilar DNA oligonucleotides (short fragments of DNA sequence) which act as primers to initiate a controlled polymerase reaction which, in turn, amplifies the genomic sequence that lies between the two oligonucleotide binding sites. The polymerase chain reaction employs a heat-stable polymerase (the Taq polymerase) which permits repeated heating and cooling of the reaction mixture. The amplification process is initiated by first heating the reaction mixture to denature (dissociate) the two complementary strands of the double stranded DNA to be amplified. Upon cooling, each single-stranded DNA oligonucleotide hybridizes to a specific region of one or the other of the complementary DNA strands, and acts as a primer for the heat-stable polymerase. The polymerase uses the oligonucleotide primers as starting points for the elongation of a DNA molecule complementary to the template DNA molecule to which each primer is hybridized. Each of the elongating DNA chains grows towards and beyond the distal primer site of the other template strand. By the end of the first cycle two double stranded copies of the intervening genomic sequence lying between the primer binding sites are generated. The cycle is repeated manyfold, exponentially doubling the number of copies each time. In this fashion even a single copy of a specific DNA sequence can be amplified to detectable levels in a relatively short period of time.
The polymerase chain reaction primer selection is limited by three factors. First, the two oligonucleotides must be complementary to sequences found in the template DNA in order for the oligonucleotides to hybridize to the template DNA. Without this initial hybridization step there would be no primer available for the DNA polymerase to use to initiate elongation, and no copy of the DNA sequence could be made. Second, the primers should hybridize to discrete and unique regions of the template DNA. If the primers hybridize to multiple different sites in the template sequence then the initiation site for elongation, and the DNA copy produced, would vary from cycle to cycle depending upon to which binding site the primer hybridized. Third, the two primer binding sites must not be too distant from one another. The elongation step optimally produces fragments up to approximately 2500 bases in length, and DNA sequences of greater length are amplified less efficiently or not at all. If chain elongation terminates before the distal primer site is incorporated into the sequence, the resultant incomplete DNA molecule will not participate in subsequent rounds of amplification.
While highly specific and unique oligonucleotide primers are desirable to eliminate multiple site hybridization in DNA from a single particular source, such restrictions require that separate primer pairs must be developed for each type of DNA to be probed. For example, an oligonucleotide primer pair that hybridizes to sites in the gag gene of one retrovirus may not hybridize to a similar region in the gag gene of another retrovirus. This is because of the inherent degeneracy of the genetic code (variability in the third base position of each codon), genetic variability from virus to virus, and the highly mutable character of the viral genome. Accordingly, either a mix of the appropriate primers must be used simultaneously in a single test, or else a series of separate tests, each using different primer pairs, must be conducted to screen a biological sample, such as a human patient sample, for the presence of viral gene sequences.
In order to be able to screen a biological sample for the presence of multiple retroviruses in a single test, using a single set of oligonucleotides, "universal retroviral primers" have been developed. Universal primers are degenerate in nature such that they will hybridize to, and serve as primers for the elongation of, functionally identical DNA sequences that are disparate at the actual base sequence level but which share sequence similarity in given regions. For example, even though the actual base sequences of retroviral gag genes are not exactly the same from strain to strain, most codons within conserved regions of the DNA sequence code for the same amino acid. Further, codons are degenerate in the third base position, creating further non-identity of DNA sequence from virus to virus. The majority of codons, and thus the majority of the DNA gene sequence, will be the same from strain to strain, and degenerate primers can be designed that take advantage of this feature such that they will hybridize with any of a variety of similar or related gene sequences.
This approach is appropriate, however, only for related families of genes that share conserved regions of DNA sequence. Primers that are "universal" for all of the retroviruses correspond and hybridize to gene sequences that are conserved throughout all of the retroviruses. Such universal retroviral primers which are capable of detecting all retroviruses generally are known in the art. These "universal retroviral primers" are capable of detecting a wide range of retroviruses by binding to highly conserved core regions of retroviral genomes.
For example, Donehower, et al., "The Use of Primers from Highly Conserved pol Regions to Identify Uncharacterized Retroviruses by the Polymerase Chain Reaction", J. Virol Methods, 28:33-46 (1990), describe DNA oligonucleotides directed to either end of a 135 basepair region of the pol gene that is highly conserved across all of the retroviruses. Similar primers directed to the same tyrosine-methionine-aspartic acid-aspartic acid (YMDD) tetrapeptide coded at the 3' end of the pol fragment, and the same leucine-proline-glutamine-glycine (LPQG) tetrapeptide coded at the 5' end of the fragment, have been described by Mack and Sninsky, in "A Sensitive Method for the Identification of Uncharacterized Viruses Related to Known Virus Groups: Hepadnavirus Model System", Proc. Natl Acad. Sci., USA 85:6977-6981 (1988). Primers that hybridize to the sequence encoding the tyrosine-methionine-aspartic acid-aspartic acid (YMDD) tetrapeptide are designated herein as "RV I" (an abbreviation for retrovirus primer I), and primers that hybridize to the sequence encoding the leucine-proline-glutamine-glycine (LPQG) tetrapeptide are designated herein as "RV II" (an abbreviation for retrovirus primer II).
Conversely, primers designed to detect exclusively a subfamily of retroviruses, such as lentiviruses, must correspond and hybridize to gene sequences that are conserved in lentiviruses only and which are not present in non-lentiviral viruses. No such oligonucleotide primer pairs, specific for and limited to the exclusive detection of viruses classified in the lentivirus subfamily of retroviruses, are currently known.
There are at least three requirements for lentiviral-specific nucleic acid oligonucleotides. The oligonucleotide sequence must include a gene sequence that is conserved throughout the lentivirus subfamily. The lentivirus-specific oligonucleotides must not hybridize to non-lentivirus DNA sequences, i.e. they must not hybridize to sequences found in other, non-lentiviral, retroviruses. Finally, lentivirus-specific PCR primer pairs must hybridize to gene sequences that are not so far removed from one another as to be ineffective or unable to function in the polymerase chain reaction or in other similar methods for amplification and detection of nucleic acid sequences.
A variety of methods for the amplification and detection of small, single copy gene sequences recently have been, or are being, developed. General reviews of these methods have been prepared by Landegren, U., et al., Science 242:229-237 (1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990). These methods include polymerase chain reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase hybridization, Q.beta. bacteriophage replicase, transcription-based amplification system (TAS), genomic amplification with transcript sequencing (GAWTS), nucleic acid sequence-based amplification (NASBA) and in situ hybridization. RNA oligonucleotides can be utilized in systems such as the Q.beta. replicase amplification system to detect lentiviral genomic sequences present in test samples. While PCR and other amplification technology is relatively straightforward and simple, the difficult task is the identification and selection of viral sequences that are unique to and specific for the particular subfamily of viruses to be detected.
Detection and diagnostic kits using lentiviral-specific nucleic acid oligonucleotides would be useful in specifically identifying lentiviral infections. The diagnostic kits currently available are only able to distinguish individual or specific viruses such as human immunodeficiency virus (HIV), not the viral subfamily involved. Lentiviral-specific nucleic acid oligonucleotides would also be useful in identifying and characterizing new and unknown lentiviral strains. This is a particularly important feature since HIV and other lentiviruses are highly mutable. For example, such oligonucleotides would be useful in testing individuals who have diseases clinically suggestive of HIV infection or AIDS, but who are HIV-negative by conventional tests. In addition, such oligonucleotides would provide independent confirmation of HIV-positive results derived from conventional tests. Such oligonucleotides would also be useful in detecting lentiviruses of other species, such as feline immunodeficiency virus, and lentiviral-like cellular sequences.
It is therefore an object of the present invention to provide nucleic acid oligonucleotide probes that are specific for lentiviruses but will not hybridize to retroviruses outside of the lentivirus subfamily.
It is a further object of the present invention to provide degenerate nucleic acid oligonucleotide probes that are specific for lentiviruses but will not hybridize to retroviruses outside of the lentivirus subfamily.
It is a further object of the present invention to provide RNA anti-sense probes that block or regulate lentiviral gene expression and can be used therapeutically to terminate lentiviral transcription, translation, or replication.
It is a further object of the present invention to provide DNA or RNA oligonucleotide primer pairs that hybridize to lentiviral-specific regions of the pol gene such that the DNA or RNA oligonucleotides act as effective primers in DNA or RNA amplification systems.
It is a further object of the present invention to provide DNA oligonucleotide primer pairs that hybridize to lentiviral-specific regions of the pol gene such that the DNA oligonucleotides act as effective primers in DNA amplification systems such as the polymerase chain reaction and similar techniques.
It is yet another object of the present invention to provide RNA oligonucleotides which hybridize to lentiviral-specific regions of the pol gene such that the RNA oligonucleotides act as probes in RNA amplification systems such as the Q.beta. replicase reaction and similar techniques.
It is still another object of the present invention to provide a method for rapid and specific detection of lentiviral gene sequences in biological materials.